Ceramide glycosylation determines the stemness of cancer stem cells Drug resistance with disseminated metastases is a cause of cancer death; in breast cancer, it ultimately claims approximately 40,500 American women per year. Tumors often progress rapidly once treatments fail, and enrichment of these tumors with cancer stem cells generally correlates with poor patient prognosis. Thus, cancer stem cells that initiate tumorigenesis and drive tumor progression might be a root cause of chemotherapy failure. Cancer stem cells are distinguished from normal adult stem cells by their tumorous behaviors. It is well known that anticancer drugs can kill normal stem cells, such as bone marrow stem cells-a source of severe side-effects; in contrast, however, some evidence suggests that anticancer drugs might act to expand cancer stem cell fractions or numbers. Our long-term goal is to understand how cancer stem cells can be specifically manipulated for preventive and therapeutic purposes. The objective of the research proposed herein is to determine how selected anticancer drugs affect breast cancer stem cell (BCSC) populations during the course of chemotherapy. It has been reported that glucosylceramide synthase (GCS) is overexpressed in metastatic breast cancers, and is a risk factor predicting poor chemotherapy response. Embryonic stem cells crucially require ceramide glycosylation catalyzed by GCS in order to retain their pluripotency. Little is known, though, concerning the possible involvement of GCS in regulating cancer stem cells. Our hypothesis is that ceramide glycosylation by GCS is a key step enabling tumor enrichment with BCSCs, and that enrichment with cancer stem cells drives rapid tumor progression and metastasis once chemotherapy fails. This application is formulated from our own preliminary data and previously published work, in which it was found that GCS is over-expressed in metastatic breast cancer, and that silencing of GCS was able to reverse drug resistance as well as prevent BCSC enrichment. We will: (1) identify the effects of anticancer drugs on enrichment of BCSCs and apoptosis of normal stem cells; (2) determine GCS-catalyzed ceramide glycosylation is a key process governing the accumulation of BCSCs and apoptosis of normal stem cells; and if so, (3) determine the signaling pathway(s) by which ceramide glycosylation promotes BCSC formation. Findings from in-vitro studies will be further explored in vivo, in orthotopic tumors exposed to anticancer drugs, employing gene silencing and overexpression strategies, and focusing on proposed role(s) of GCS in increasing the formation of BCSCs vs. contrasting apoptotic effects on bone marrow stem cells and normal mammary epithelial stem cells. Results will be further corroborated via studies of tumor and bone marrow samples from patients undergoing chemotherapy. Lastly, the therapeutic efficacy of disrupting ceramide glycosylation by an antisense oligonucleotide or a small molecule in order to effect reductions in BCSCs in orthotopic tumors will be examined, wherein success will lend validity to this unique approach for selectively eliminating cancer stem cells via deletion of key glycosphingolipids. Completion of the proposed studies will introduce new concepts and advanced technologies in cancer stem cells and drug discovery to students who participate, thus greatly enhancing their biomedical sciences education.